Industrial alkaline protease from shipworm bacterium

ABSTRACT

A protease has been isolated from a symbiotic bacterium found in the gland of Deshayes of the marine shipworm. The protease remains active over the pH range of about 4-12, exhibits salt tolerance up to 3M sodium chloride, retains a high level of activity above 50° C. for at least 60 min, and is stimulated by oxidizing agents, particularly perborate. The properties of this protease suggest widespread utility in detergents and other low-temperature industrial applications.

This is a continuation of application Ser. No. 07/880,912, filed May 12,1992, now U.S. Pat. No. 5,312,749.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates to a novel protease which has been isolated forma bacterial symbiont of the marine shipworm. The protease is uniquelycharacterized by stability over a broad pH range as well as tolerance toa variety of chemical environments. These properties render the proteaseuseful in detergents and other low temperature industrial applications.

2. Description of the Prior Art

Proteolytic enzymes are conventionally used in detergent compositions,particularly in laundry detergents. One such enzyme commonly used issubtilisin. This enzyme is produced by Bacillus subtilis and is aneffective stain removing agent. Proteolytic enzymes derived from avariety of other Bacillus species have been disclosed by Aunstrup et al.in U.S Pat. No. 3,723,250. The sources of these species were largelysoil samples and manures, and the enzymes were selected for optimalproteolytic activity against hemoglobin at pH above about 9.

Hora (Canadian Patent 1,247,025) recognizes the enzyme stabilityproblems in enzymatic laundry detergents containing peroxideprogenitors. Peroxy-type bleaching agents of concern are alkali metalpersulphates, perphosphates, perborates, as well as alkali metal andalkaline earth metal salts of organic peracids. Hora discovered that thestorage stability of these compositions could be improved by theinclusion of an alkali metal metaborate.

Another factor affecting the stability of proteolytic enzymes is pH. InU.S. Pat. No. 3,840,433, Aunstrup et al. identify a group of proteasesproduced by the cultivation of Bacillus alcalophilus. These enzymes areuseful in the dehairing of hides, and can withstand the highly alkalinepH of a saturated lime solution or of a soda ash solution. Aunstrup etal. contend that the B. alcalophilus proteases exhibit maximum activityagainst hemoglobin at pH 12 and 25° C. and also at pH 10.1 and 60° C.

Hellgren et al. (U.S. Pat. Nos. 4,801,451 and 4,963,491) teach enzymepreparations from animals of the order Euphausiaceae as being useful incleaning compositions for degrading and removing contaminants ofbiological origin. At optimum temperatures of 30°-55° C., thesepreparations display proteolytic activity in the pH range 5-10.

In U.S. Pat. No. 4,865,983, Durham et al. teach the use of proteasesfrom Vibrio species in cleaning compositions, including laundrydetergents, automatic dishwasher detergents, laundry bleaches andpresoaks. These proteases are characterized by a high proteolyticactivity, stability over wide pH and temperature ranges, and stabilityto oxidizing agents, including chlorine bleaches.

Lad et al. (U.S. Pat. No. 4,749,511) relate to contact lens cleaningsolutions which are comprised of general-purpose proteases incombination with the endoproteinase lys-C for removing lysozyme fromlens surfaces. The function of the endopeptidase is to specificallycleave at the carboxy side of lysine residues and expose susceptiblepeptide bonds of lysozyme to the protease without concomitantinactivation of the protease. The cleaning solutions of Lad et al.include additional components which aid in the overall lysozymedegradation and solution stability.

In general, enzymes ideal for use in detergents and other cleansingcompositions should possess a high level of activity on proteinaceouscontaminants over a broad pH range and over a broad temperature range.They should also be stable in the presence of oxidizing agents,bleaches, surfactants and other additives commonly used in detergents.Some detergent applications present less stringent conditions thanothers and do not require enzymes having the optimum range oftolerances. For example, enzymes for room temperature presoaks andcontact lens solutions typically do not require the same temperaturestability range continues to extend the conditions of activity andstability of proteolytic enzymes for all applications.

SUMMARY OF THE INVENTION

We have now discovered a novel protease which remains active over the pHrange of about 4-12, exhibits salt tolerance up to saturated NaCl (>3M)sodium chloride, retains a high level of activity above 50° C. for atleast 60 min, and is stable to cleansing composition constituents,including oxidizing agents and complexing agents. In fact, the novelprotease appears to be unique for its characteristic of proteaseactivity stimulation by oxidizing agents, particularly perborate. Thisenzyme was isolated from a symbiotic bacterium found in the gland ofDeshayes of the marine shipworm, Psiloteredo healdi. Its propertiessuggest widespread utility in cleansing compositions and otherlow-temperature industrial applications.

In accordance with this discovery, it is an object of the invention toprovide a novel proteolytic enzyme in substantially pure form.

It is also an object of the invention to characterize the subject enzymeregarding its useful chemical and physical properties for industrialapplications.

It is a further object of the invention to demonstrate the utility ofthe subject protease in a variety of cleansing compositions, such aslaundry detergents and contact lens cleaning solutions.

These and other objects of the invention will become readily apparentfrom the ensuing description

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A, 1B, and 1C are plots showing the effects of temperature, ph,and salinity respectively on the activity and stability of the purifiedalkaline protease of the invention.

FIG. 2 is a pair of plots showing the effects of NaBO₃ and H₂ O₂ on therelative activity of the purified protease of the invention.

DETAILED DESCRIPTION OF THE INVENTION

The source of the protease of the invention is a symbiotic bacteriumfound as a pure culture in the Gland of Deshayes in many species ofwood-boring shipworms which make up a group of bivalve molluscs known asthe Teredinidae. As explained by Waterbury et al. in U.S. Pat. No.4,861,721, herein incorporated by reference, the Gland of Deshayes isunique to the Teredinidae and has historically been associated with theproduction of cellulolytic enzymes responsible for the wood-boringcapability of the mollusc. Bacteria present in this gland were firstisolated and reported by Waterbury et al. as being both cellulolytic andnitrogen-fixing. The patent further indicates that bacterial isolatesfrom 15 species within the family Teredinidae were so similar that theylikely represent a single species. Also, the fact that the bacterium wassuccessfully isolated from every species of shipworm examined suggeststhat the bacterium is found in all species of Teredinidae. Accordingly,it is a reasonable expectation that the extracellular protease of theinvention can be isolated from the Gland of Deshayes bacterium from allshipworm species. The species of shipworm used as the source of thebacterium for the isolation disclosed herein was Psiloteredo healdi.This bacterium is available from the American Type Culture Collection,Rockville, Md., under the Accession Number ATCC 39867.

The shipworm bacterium is readily cultivated on cellulose or othersuitable carbon source, preferably, but not necessarily, supplementedwith an inorganic nitrogen source. A suitable cultivation temperaturewould be in the range of 10°-35° C. and preferably 25°-30° C. Theoptimum cultivation period for a given medium and temperature can bedetermined by assaying the supernatant activity against a proteinsubstrate, such as azocasein.

In the preferred method of isolating the protease, the whole culture iscentrifuged at approximately 10⁴ g to remove cells, and the recoveredsupernatant is then subjected to ultracentrifugation at approximately10⁵ g to remove cellular debris. The ultracentrifugation supernatant isconcentrated by ultrafiltration and then diafiltered against basal salt("Amicon PM 30" hollow fiber membrane, Amicon, Danvers, Ma.), and theresulting filtrate is recovered. The diafiltration serves to separatethe protease activity from the extracellular endoglucanase activitywhich is retained in the concentrate [see Greene et al., Achieves ofBiochemistry and Biophysics, vol. 267, 334-341 (1988)]. The combinedfiltrate from the ultrafiltration and the diafiltrate are furtherconcentrated by ultrafiltration on a "PM 10" hollow fiber membrane(Amicon); and the protease activity is precipitated from theconcentrate, such as by bringing the concentrate to 65% saturation with(NH₄)₂ SO₄. The precipitate is then resuspended and fractionated by gelpermeation chromatography. Of course, it is understood that well-knownalternatives could be substituted for the various separation steps andthat other variations of this scheme would also serve to isolate theproteolytic enzymes.

The apparent molecular mass of the protease of the invention isdependent upon the procedure by which it is determined. Analysis bySDS-PAGE yields a relative molecular mass of 36,000 daltons. Whendetermined by serial gel permeation HPLC, the mass is 17,600±300daltons. The rate zonal sucrose density-gradient centrifugation methodyields a mass of 46,000±2,000 daltons.

While not desiring to be bound by any particular theory, there is aplausible explanation for the large discrepancy in the molecular massvalues obtained by the various methods. The low mass (17,600 daltons)obtained by gel permeation HPLC may have resulted from nonspecificinteraction of the protein with the column resin, retarding its elution.However, varying the ionic strength of the eluent, addition of detergentor addition of exogenous protein did not significantly alter the elutionprofile of the protease. A molecular mass of 17,600 is also in goodagreement with the nominal molecular weight cutoff values of theultrafiltration membranes used in the purification scheme described inExample 2. The molecular mass of 36,000 daltons determined by SDS-PAGEand the mass of 46,000 daltons determined by zonal density-gradientcentrifugation suggests that the native protease is a dense, tightlyfolded molecule. For such an enzyme, sedimentation behavior, which isstrongly and directly dependent on protein density, would be expected toyield an anomalously high apparent molecular mass relative to standardproteins. Conversely, estimates of molecular mass by HPLC and thosesuggested by ultrafiltration behavior would be anomalously low, as bothdeterminations depend primarily on the radii of molecules. A denser,more tightly folded molecule should possess a smaller radius incomparison to equivalent molecular mass standards. The molecular massdetermined by SDS-PAGE should be close to the actual molecular mass ofthe protease. Other characteristics of the protease are described infurther detail in Example 3.

The activity of the protease of the invention remains unaffected byseveral compounds known to inhibit various other proteases. For example,sodium perborate, the metal-ion chelators EDTA and Neocuproine HCl, andthe cysteine protease inhibitor iodoacetamide have little effect on theenzyme's activity. Phenylmethylsulfonyl fluoride (PMSF), which reactswith the hydroxyl moieties of active site serines in serine proteases,does not inhibit activity. However, tosyl-1-lysine chloromethyl ketone(TLCK), which inhibits serine proteases with trypsin-like activity atthe reactive site histidine residue, significantly reduces the activityat low concentrations and abolishes it at high concentrations. Inaddition, the enzyme is unaffected by tosylamide phenylethylchloromethyl ketone (TPCK), a chymotrypsin inhibitor, or byε-Amine-n-capronic acid, a plasmin inhibitor.

The high specific activity of the purified protease coupled with itsinsensitivity to chelating agents and sodium perborate, as well as itsstability to an extremely broad range of pH values and ionic strengthsrenders it useful for a multitude of commercial applications. Withoutlimitation thereto, it is envisioned that the enzyme would be useful inlow-temperature laundry detergents, presoaks, household cleaners, bodycleansers, contact lens cleaning solutions, dehairing and leathertanning agents, as well as in methods for raw silk cleaning (degumming),silver recovery from photographic film, haze elimination from beers andother beverages, and gelatin manufacturing.

Compositions comprising the protease of the invention may also compriseingredients and additives conventionally used for the desiredapplication, provided that they do not substantially interfere with theactivity of the protease. For example, detergent formulations are likelyto include surfactants, soaps, detergents, bleaching agents, builders,soil-suspending agents, pH buffers, buffers, lipases, amylases,cellulases, fragrances, optical brighteners, softening agents,stabilizers, carriers, and the like. Detailed illustrations of suchadditives are given in Durham et al., supra, herein incorporated byreference. Similarly, dehairing solutions may contain lime, causticsoda, or other alkali such as described in Aunstrup et al., supra,herein incorporated by reference, Hellegren et al., supra, hereinincorporated by reference, teach various additives for body cleansers,such as antimicrobials, surfactants, pharmaceutically acceptablecarriers, gel formers, etc. As described in Lad et al., supra, hereinincorporated by reference, contact lens cleaning solutions may containvarious detergents, surfactants, buffers, stabilizers, binders,lubricants, and the like.

Any composition or formulation having as its principal function thecleansing or decontamination of an object, body, or material is definedherein as a "cleansing composition". Cleansing compositions embodyingthe protease of the invention will typically include one or morenonproteolytic chemical cleansing agent which complements theproteolytic activity in producing the desired cleansing action. Examplesof such agents, include soaps, detergents, surfactants, and otherenzymes.

The enzyme-containing compositions of the invention should contain anappropriate amount of enzyme for achieving the desired end result. Forexample in laundry detergent compositions, the level of enzyme should besufficient to degrade at least most of the proteinaceous contaminantslikely to be present in soiled clothes. It is understood that the otherlaundry detergent components discussed above would also play a role inthe cleansing action of the composition. Therefore, an effective amountof enzyme would be determined by the nature and amount of otherdetergent additives in the formulation, as well as by the expectedchemical and physical conditions of use. Similar considerations wouldapply to the selected enzyme level for compositions intended for otherapplications. In general, the level of proteolytic activity for anygiven compositions will be in the range of about 10 to 15 enzyme unitsper milliliter of solution. An enzyme unit is defined as 1 μg ofazoprotein solubilized per hour.

It is also contemplated within the scope of the invention to include theendoglucanase produced by the marine shipworm bacterium as a componentof the various compositions described above. This can be accomplished byemploying a bacterial culture fraction which comprises both the proteaseand the endoglucanase, or by recombining these enzymes after separation.As discussed in Griffin et al. [Biochemical and Biophysical ResearchCommunications, vol. 144, 143-151 (1987)] and in Greene et al. [Archivesof Biochemistry and Biophysics, vol. 267, 334-341 (1988)], theendoglucanase produced by the marine shipworm bacterium is useful forinitiating cellulose degradation and would therefore be a desirablecomponent in many of the detergent compositions previously discussed.

This invention is further illustrated by the following examples.

EXAMPLE 1 Cell Culture

The bacterial strain (Woods Hole Oceanographic Institution strain numberT8301) isolated from Psiloteredo healdi shipworm, was a gift of Dr. JohnWaterbury. Cultures (1 liter) were grown aerobically in 3-liter Fernbachflasks on basal salt medium supplemented with 1% Sigmacell type 50 as acarbon source and 0.10% NH₄ Cl as a nitrogen source. The basal mediumcontained the following, per liter of deionized H₂ O:NaCl₂, 18.8 g; KCl,0.4 g; MgSO₄.7H₂ O, 1.9 g; MgCl₂.6H₂ O, 1.5 g; CaCl₂.2H₂ O, 0.4 g; HEPES(N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid), 4.9 g; solutionA, 10 ml; trace metal solution, 1 ml. Solution A consisted of thefollowing (per liter of H₂ O): K₂ HPO₄.3H₂ O, 2.0 g; Na₂ CO₃, 1.0 g;sodium citrate, 0.4 g; Fe₂ (SO₄)₃, 0.3 g; EDTA, 0.05 g. Trace metalsolution consisted of the following (per liter of H₂ O): H₃ BO₃, 2.9 g;MnCl₂.4H₂ O, 1.8 g; ZnSO₄.7H₂ O, 0.2 g; Na₂ MoO₄.2H₂ O, 0.04 g;CoSO₄.7H₂ O, 0.05 g; CuSO₄.5H₂ O, 0.08 g. The pH of the basal medium wasadjusted to 8.0 with NaOH. The standard growth temperature was 30° C.

EXAMPLE 2 Protease Purification

Cultures (4×1 liter) were harvested after 10 days of growth bycentrifugation at 15,000 g for 30 min, 4° C. The cell pellet wasdiscarded and the supernatant was ultracentrifuged at 100,000 g at 4° C.for 30 min. The clear, membranous pellet obtained fromultracentrifugation was also discarded. The supernatant was concentratedtenfold by ultrafiltration ("Amicon PM 30" hollow fiber membrane,Amicon, Danvers, Ma.) and then diafiltered with six volumes of distilledwater. The combined filtrate and diafiltrate were concentrated to 400 mlby ultrafiltration on an "Amicon PM 10" hollow fiber membrane. Theprotease activity of the "PM 10" retentate was precipitated by bringingthe concentrate to 65% saturation with (NH₄)₂ SO₄ and collecting theprecipitate by centrifugation for 10 min at 10,000 g at 4° C.

The precipitate was resuspended in aqueous 0.1M NaCl containing 20 mMHEPES buffer at pH 7.0. Aliquots of the solution (200 μl) were appliedto a HPLC system fitted in series with a "Bio-sil Sec.250" and a"Bio-sil Sec. 125" column (Bio-Rad Laboratories, Richmond, Ca.). The gelpermeation system was equilibrated and eluted with aqueous 0.10M NaClcontaining 20 nM HEPES buffer at pH 7.0. Fractions containing proteaseactivity were immediately frozen at -72° C. and stored at -20° C. untilused.

The protease purification was monitored by assaying for proteolyticactivity and protein concentration after each step. Proteolytic activitywas determined in duplicate assays employing azocasein for thesubstrate, as described by Cotta et al. [Applied EnvironmentalMicrobiology, vol. 52, 51-58 (1986)]. Units represent μg of azocaseindigested per hour. Protein concentrations were determined by the methodof Bradford [Analytical Biochemistry, vol. 5, 271-283 (1976)] withbovine serum albumin as a standard.

The results of the purification of a protease from 4 liters of cell-freeculture medium are summarized in Table I. Ultracentrifugation ofcell-free medium removed about half of the protein, while most of theprotease activity remained in solution. Further purification wasachieved by ultrafiltration as activity passed through a "PM 30"membrane, but was retained by a "PM 10" membrane. This procedureincreased the specific activity of the preparation by 1.6-fold, but onlyafter a significant loss in total yield. Much of the loss in yieldresulted from activity retention by the "PM 30" membrane. Such activitycould not be diafiltered through the "PM 30" membrane. Afterconcentration on the "PM 10" membrane, the protease activity wasprecipitation with 65% saturated ammonium sulfate and could be sortedindefinitely at -20° C. Gel permeation chromatography allowedpurification of the protease to homogeneity as determined by SDS-PAGE.Typically, this purification scheme allowed for a 23-fold increase inspecific activity with a 21% yield. It should be noted that thesenumbers are subject to a certain degree of variability due tocompetitive inhibition of the azocasein assay by endogenous bacterialprotein, as well as autohydrolysis of the enzyme itself.

EXAMPLE 3 Physical Characterization

Solutions of the purified protease from Example 2 at appropriate proteinconcentrations and in proper solvents for physical characterization wereprepared from relatively dilute gel permeation fractions by centrifugalultrafiltration. This process involved concentrating and washing withappropriate solvents in "Amicon Centricon-10Microconcentrators" (amicon1989 Operating Instructions Centricon Microconcentrators for smallvolume concentration, publication No. 1-259E).

Molecular Mass

The native molecular mass of the protease was determined by gelpermation chromatography and by rate zonal sucrose density-gradientcentrifugation; the denatured molecular mass of the protease wasdetermined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE).

The determination by gel permeation chromatography used the HPLC systemand mobile phase described above for protease purification. Protein wasdetected by absorbance at 280 nm and protease activity by the assaypreviously described. The relative molecular mass of the enzynme wasinterpolated from a plot of log molecular mass versus retention time forstandard proteins. The average mass value of five determinations by thismethod was 17,600±300 daltons.

For rate zonal sucrose density-gradient centrifugation, aliquots (0.1ml) of purified protease and marker protein solutions were layered on4.0 ml of a 5-20% linear sucrose gradient in 4.4 ml polyallomercentrifuge tubes. All solutions were buffered at pH 7.0 with 20 mM HEPESbuffer containing 0.1M NaCl. The tubes were centrifuged 17.5 hr at50,000 rpm in a swinging bucket rotor (SW 56 Ti, Beckman Instruments,Inc., Palo Alto, Ca.) at 2° C. After centrifugation, the contents of thetubes were analyzed for absorbance at 254 nm and fractionated using an"Isco Density Gradient Fractionator". The fractions (0.25 ml) wereassayed for protease activity. Distance sedimented by the markerproteins [Ribonuclease A (13,700) chymotrypsinogen A (25,000), ovalbumin(45,000), Aldoloase (158,000)] was calculated from the position ofmaximum absorption at 254 nm. Distance sedimented by the protease wascalculated from the position of maximum protease activity. The relativemolecular mass of the protease was interpolated from a plot of logmolecular mass versus log distance sedimented for the marker proteins.The average value for three determinations was 46,000±2000.

Determination of denatured molecular mass by SDS-PAGE was performed bythe method of Laemmli [Nature, vol. 227, 680-685 (1970)] using a"PhastSystem" (Pharmacia, Piscataway, N.J.) and a precast continuous10-15% polyacrylamide gradient gel with 2% crosslinking. Bands weredetected by silver staining. The relative molecular mass of the proteasewas interpolated from a plot of log molecular mass versus relativemobility for standard proteins. A single band with a relative molecularmass of 36,000 daltons was obtained.

Isoelectric Point

Nondenaturing isolectric focusing was also performed with the"PhastSystem" using "PhastGel IEF 5-8", a homogenous polyacrylamide gelcontaining "Pharmalyte" carrier ampholytes that generate a linear pHgradient from pH 5.85 to pH 10.25 during electrophoresis. Bands weredetected by silver staining. The pI of the protease was interpolatedfrom a plot of PI for standard proteins versus their distance from thecathode after reaching equilibrium position. This method revealed analkaline doublet at pH 8.53 and pH 8.63 indicating proteinmicroheterogeneity.

Proteolytic Activity

Proteolytic activity was detected by blotting identically run butunstained IEP "PhastGels" onto a Bio-Rad agar diffusion plate (10 mmthick 1% agar gel containing bovine casein in Tris-buffered saline, pH7.4 ), for 16 hr at ambient temperature. Following removal of the"PhastGel", the casein agar was layered with 5% acetic acid in 10%aqueous ethanol to precipitate undigested bovine casein. A clearingzone, indicative of proteolytic activity, became readily apparent andwas centered around the doublet.

Carbohydrate Content

Carbohydrate content of the protease was determined by thephenol-sulfuric acid method of Dubois et al. [Analytical Chemistry, vol.28, 350-356 (1956)]. Sufficient frozen active gel permeation fractionsto contain >100 μg of protease protein were concentrated with "AmiconCentricon-10" microconcentrators to 0.5 ml and were washed with sixvolumes of distilled water to remove free sugars. The sample was thenassayed for carbohydrate using glucose as the standard. Within thedetection limits of the phenol-sulfuric acid assay (1%), no carbohydrateappeared to be associated with the enzyme.

Temperature, pH, Salinity Dependence, and Stability

As shown in FIGS. 1A, 1B and C of temperature, pH, and salinityrespectively on the activity of the purified protease were determined.The stability (□) of the protease against each factor was determined bypreincubation at the indicated temperatures, pHs, and salinities for 60min and then reacting with azocasein in standard assays. The standardazoprotein assay was reacted 3 hr at 25° C., pH 6.8, and 0.10M NaCl. Theactivity dependence (∘) of the protease on each factor was determined byadjusting the standard azocasein assay to the temperature, pH, orsalinity indicated. Maximum relative activity corresponds to 150units/ml.

Maximum activity was observed at 42° C., pH 9.0, and 0.20M NaCl. Theenzyme was unstable at temperatures exceeding 40° C., below 0.10M NaCl,and at pH values below 3.0 or above 11.9. Activity reduction above 0.40MNaCl could have related to physical changes in the assay substrate. Thatis, visible precipitation of the azocasein occurred at high salinities.The loss of protease activity noted at NaCl below 0.10M was accompaniedby significant aggregation. In water, the M_(r) of the major protein isnear 168,000 daltons (gel permeation HPLC analysis) and only about 6% ofthe original activity remained. Such aggregation could be prevented andnearly half of the protease activity retained by including a substrate(0.02% BSA) in the solvent. Loss of activity that occurred above 42° C.(FIG. 1A) was not stabilized significantly by added salt or substrate.

The purified enzyme had a specific activity of 11,240 proteolyticunits/mg measured in the standard assay (25° C. pH 6.8, and 0.10M NaCl).At optimum conditions (42° C., pH 9.0, and 0.20M NaCl), this value wasincreased to 65,840 units/mg. The enzyme was stable under theseconditions for at least 12 hr. However, under conditions of instability(temperatures greater than 42° C., salinities below 0.10M, and pH above11.9 and below 3.0), the activity half-life was less than 30 min.

EXAMPLE 4

Inhibition of Protease Activity

Several compounds known to inhibit various proteases were examined fortheir effect on the activity of the purified enzyme from Example 2(Table II). The protease was incubated prior to assay for 1 hr at 25° C.(except where noted) in buffer containing the inhibitor at theconcentrations shown in the Table. All assays were measured at 42° C.and pH 9.0 in 0.2M NaCl buffered with 100 mM bis Tris propane. Onehundred percent activity with no inhibitor present was 154 units/ml.

Of particular significance from the data presented in Table II is theapparent enhancement of protease activity produced by the sodiumperborate tetrahydrate over a range of temperatures. The effect ofoxidants on protease activity was further investigated in Example 5,below.

EXAMPLE 5 Effect of Oxidants on Protease Activity

The effects of hydrogen peroxide and sodium borate on the activity ofthe alkaline protease were investigated. Samples containing 224 units ofprotease were incubated in the presence of the oxidant for 3 hr at 25°C. Bis Tris propane (20 mM) in 0.15M NaCl was used to buffer the systemat pH 9.0. The relative activity of azocasein is plotted versus theweight percent of hydrogen peroxide (∘) or NaBO₃ (•) in FIG. 2.

EXAMPLE 6 Amino Acid Analysis and Sequencing

Sequencing and amino acid composition of the protease was performed byautomated Edman degradation on an Applied Biosystems (San Jose, Ca.)477A pulsed-liquid phase sequencer by Dr. Saw Kyin (University ofIllinois Biotechnology Center).

Table III presents the amino acid composition of the purified proteasebased on a relative molecular weight of 36,000 daltons. As suggested bythe high isoelectric point, the enzyme was rather rich in basicresidues. The basic residues, histidine, arginine, and lysine,represented 10% of the protein hydrolysate. The sequence obtained forthe first 10 residues of the enzyme wasIle--val--try--pro--arg--val/ala--gly--met--ser (SEQ ID NO 1). Withinthe error limits of the assay, residue number 6 was uncertain andassigned either valine or tyrosine was valine being more probable.

EXAMPLE 7 Effect of Protease Presoaking on Cleansing Power of Detergent

Washing experiments were carried out using swatches of EMPA 116 testcloth uniformly soiled with blood, mil, and carbon black.

The detergent used in these experiments was AATCC Standard detergent 124without brightener having the following composition:

    ______________________________________                                        Linear alkysulfonate-sodium salt (LAS)                                                                  14.00%                                              Alcohol ethoxylate        2.30%                                               Soap-high molecular mass  2.50%                                               Sodium triplyphosphate    48.00%                                              Carbonate                 0.00%                                               Alumino silicate solids   0.00%                                               Sodium silicate           9.70%                                               (SiO.sub.2 /Na.sub.2 O = 2.0:1 ratio)                                         Sodium sulfate            15.40%                                              Carboxymethylcellulose (CMC)                                                                            0.25%                                               Sodium polyacrylate       0.00%                                               Moisture and miscellaneous                                                                              7.85%                                               Brightener 15             0.00%                                               ______________________________________                                    

Other conditions were as follows:

    ______________________________________                                        Water hardness       5 ppm                                                    Fabric to water ratio                                                                              1:40                                                     Wash time            30 min                                                   pH                   9.0                                                      Detergent concentration                                                                            0.40% (wt basis)                                         ______________________________________                                    

For this example, the protease was used as a presoak in the followingprocess. At zero time, 10 ml of enzyme solution at pH 9.0 containing1200 units of azocasein activity was pipetted into a 150-ml beakercontaining 90 ml of tap water or sea water which was equilibrated andthermostatted at 50° C. Concurrently, 2.5 g of the test fabric was addedto the contents of the beaker and allowed to soak for 30 min. Controlswere run using tap water and sea water presoaks without protease as wellas elimination of the presoak step altogether. At the end of the presoakperiod, the fabric was lifted out of the beaker and placed into another150-ml beaker containing 90 ml of wash solution (tap water withdetergent) which was equilibrated and thermostatted at 50° C. Every 4min the contents of the beaker were agitated for 10 sec with a glassrod. After 30 min the was solution was drained, and the test swatcheswere rinsed in running tap water for 10 min. They were subsequentlyair-dried and ironed. The remission of the ironed swatches was measuredin a LI-COR Portable Spectraradiometer equipped with an integratingsphere at 480 nm. Remission values were standardized against BaSO₄. Thereported remissions indicate average values from six randomly selectedswatch locations. The results are reported in Table IV.

EXAMPLE 8

The procedure of Example 7 was repeated except that the enzyme solutionwas combined with the wash solution prepared from either tap water orsea water, and the presoak step was eliminated. Controls included tapwater and sea water detergent solutions without protease or withautoclaved protease. The results are reported in Table V.

EXAMPLE 9

The procedure of Example 8 was repeated using tap water plus enzyme asthe wash solution at four different temperatures. The results arereported in Table VI.

It is understood that the foregoing detailed description is given merelyby way of illustration and that modification and variations may be madetherein without departing from the spirit and scope of the invention.

                                      TABLE I                                     __________________________________________________________________________    Summary of Purification of Alkaline Protease from Shipworm Bacterium                        Total protein                                                                        Total activity                                                                       Specific activity                                                                     Fold  Yield                               Fraction      (mg)   (U · 10.sup.3)*                                                             [(U/mg) · 10.sup.3 ]*                                                        purification                                                                        (%)                                 __________________________________________________________________________    Whole culture --     384.0  --      --    --                                  Centrifuge supernatant                                                                      760    369.5  0.49    1.0   100                                 Ultracentrifuge supernatant                                                                 344    277.8  0.81    1.6   75                                  "PM 10" retentate                                                                            34     47.2  1.39    2.8   13                                  (NH.sub.4).sub.2 SO.sub.4 precipitate                                                        28     39.6  1.41    2.9   11                                  Gel permeation HPLC                                                                          7      78.7  11.24   22.9  21                                  __________________________________________________________________________     *Units (U) are micrograms of azoprotein solubilized per hour.            

                  TABLE II                                                        ______________________________________                                        Inhibition of Alkaline Protease                                               Inhibitor                 Conc. of  Activity                                  (class)     Inhibitor     inhibitor (%)                                       ______________________________________                                        Serine and cysteine                                                                       Phenylmethane-                                                                              0.1    mM   93                                      protease inhibitor                                                                        sulphonyl fluoride                                                                          1      mM   98                                                                10     mM   102                                     Trypsin, plasmin,                                                                         TLCK          0.1    mM   47                                      thrombin, and             1      mM   20                                      cysteine protease         10     mM    7                                      inhibitors                                                                    Chymotrypsin and                                                                          TPCK          0.1    mM   94                                      cysteine protease         1      mM   99                                      inhibitor                 10     mM   100                                     Plasmin and trypsin                                                                       ε-Amino-n-caproic                                                                   100    μg                                                                              101                                     protease inhibitor                                                                        acid          500    μg                                                                              99                                      Cysteine protease                                                                         Iodoacetamide 0.1    mM   102                                     inhibitor                 1      mM   102                                                               10     mM   100                                     Heavy-metal ions                                                                          CuCl.sub.2    0.1    mM   97                                                                10     mM   94                                                  HgCl.sub.2    0.1    mM   66                                                                10     mM    8                                      Metal-ion chelators                                                                       EDTA (disodium                                                                              10     mM   102                                                 salt) Neocuproine                                                                           10     mM   99                                                  HCl                                                               Bleaching agent                                                                           Sodium perborate                                                                            0.1%                                                            tetrahydrate  (25° C.)                                                                         119                                                                 (42° C.)                                                                         117                                       ______________________________________                                    

                  TABLE III                                                       ______________________________________                                        Amino Acid Composition of Alkaline Protease                                                  Molecular ratios                                                                         Nearest                                             Amino acid       Average  integer                                             ______________________________________                                        Aspartic acid/asparagine                                                                       40.59    41                                                  Glutamic acid/glutamine                                                                         2.07     2                                                  Serine           25.29    25                                                  Glycine          32.77    33                                                  Histidine         5.20     5                                                  Arginine         17.26    17                                                  Threonine        16.91    17                                                  Alanine          34.07    34                                                  Proline          10.91    11                                                  Tyrosine         16.37    16                                                  Valine           34.22    34                                                  Methionine        1.00     1                                                  Isoleucine       21.92    22                                                  Leucine          17.41    17                                                  Phenylalanine     9.32     9                                                  Lysine            6.52     6                                                  ______________________________________                                    

                                      TABLE IV                                    __________________________________________________________________________    Effect of Protease Presoaking on Cleansing Power of Detergent                            Remission of EMPA-test swatch No. 116                                                        After washing                                                        After presoaking                                                                       presoaked swatch                                    Presoak solution                                                                         Untreated                                                                           and rinsing                                                                            in tap water with detergent                         __________________________________________________________________________    None       10.1  --       20.3                                                Tap water  --    15.4     34.1                                                Tap water + protease                                                                     --    21.9     45.4                                                Sea water  --    12.5     36.0                                                Sea water + protease                                                                     --    11.2     43.0                                                __________________________________________________________________________

                  TABLE V                                                         ______________________________________                                        Effect of Protease Addition on                                                Cleansing Power of Detergent                                                                       Remission of                                                                  EMPA-test                                                                     swatch No. 116                                           Detergent wash solution                                                                            After washing                                            ______________________________________                                        Tap water            20.3                                                     Tap water + protease 34.7                                                     Tap water + autoclaved protease                                                                    21.7                                                     Sea water            14.7                                                     Sea water + protease 16.0                                                     Sea water + autoclaved protease                                                                    14.4                                                     ______________________________________                                    

                  TABLE VI                                                        ______________________________________                                        Effect of Temperature on Cleansing                                            Power of Detergent with Protease Additive                                                     Remission of EMPA-test                                        Detergent wash  Swatch No. 116                                                solution        25°                                                                           30°                                                                             40°                                                                         50°                               ______________________________________                                        Tap water       --     --       --   21.7                                     Tap water + enzyme                                                                            37.4   35.0     35.7 37.7                                     ______________________________________                                    

    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES: 1                                                  (2) INFORMATION FOR SEQ ID NO:1:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 10 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: Psiloteredo healdi                                               (C) INDIVIDUAL ISOLATE: ATCC 39867                                           (ix) FEATURE:                                                                 (A) NAME/KEY: Region                                                          (B) LOCATION: 1..10                                                           (D) OTHER INFORMATION: /note="This sequence represents                        the first 10 residues of the protease isolated in                             Example 2 "                                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                       IleValTyrProArgValAlaGlyMet Ser                                               1510                                                                      

We claim:
 1. A cleansing composition comprising a chemical cleansingagent and a protease, wherein the protease is the same as that producedby bacteria found in the gland of Deshayes of the marine shipworm in thefamily Teredinidae, has a molecular mass of about 36,000 daltons asdetermined by SDS-PAGE, has an isoelectric point of about pH 8.6, ischaracterized by the property of proteolytic activity stimulation byoxidizing agents and is present in the composition in an effectiveamount for degrading targeted proteinaceous contaminants, and whereinthe chemical cleansing agent is present in an amount which does notsubstantially interfere with the activity of the protease.
 2. Acleansing composition as described in claim 1 wherein said marineshipworm is Psiloteredo healdi.
 3. A cleansing composition as describedin claim 2 wherein said bacteria is ATCC
 39867. 4. A cleansingcomposition as described in claim 1 and further comprising an oxidizingagent.
 5. A cleansing composition as described in claim 4 wherein saidoxidizing agent is selected from the group consisting of perborates andperoxides.
 6. A cleansing composition as described in claim 1 whereinsaid chemical cleansing agent is a detergent.
 7. A cleansing compositionas described in claim 1 and further comprising an extracellularendoglucanase.
 8. A cleansing composition as described in claim 7wherein said endoglucanase is produced by bacteria associated with themarine shipworm in the family Teredinidae.